Influenza viruses belong to the family Orthomyxoviridae and are separated into types A, B and C according to antigenic differences. Influenza A and B viruses are important respiratory pathogens, although influenza A viruses are the main cause of epidemics with high mortality rate.
Influenza viruses have a segmented, single-stranded RNA genome of negative polarity. The genome of influenza A viruses consist of eight RNA molecules encoding eleven (some influenza A strains ten) protein. The viral envelope is composed of a lipid bilayer on a layer of matrix protein (M1). Embedded in the lipid bilayer are glycoproteins hemagglutinin (HA) and neuraminidase (NA), which are responsible for virus attachment and penetration into the cell, and release of progeny virus from the infected cells, respectively. Both proteins also determine the subtypes of the influenza A viruses. High mutation rates and frequent genetic reassortment due to the segmented genome, contribute to great immunological variability, particularly of the HA and NA antigens of influenza A viruses. Type B viruses do not exhibit the same degree of antigenic variation and are primarily childhood pathogens, which can occasionally cause epidemics of the same severity as type A.
The incubation time for influenza ranges from 1 to 5 days. Clinical onset is characterised by abrupt fever, headache, malaise and myalgias. Systemic symptoms usually last for 3 days. Although precise data on influenza associated deaths are not available for all countries, in the United States influenza associated deaths range between 30 and 150 per 100000 population aged over 65.
During influenza epidemics attack rates of 1%-5% are most common, but the attack rate may reach 40%-50%. Economic impact of influenza epidemic or even pandemic can therefore be tremendous. Vaccination was recognised as the best approach to limit the epidemics and its harmful effects.
Since introduction in 1940s vaccines based on virus material cultured in chicken eggs have been found to be clearly effective against influenza infection and have resulted in a significant fall in the mortality rate of high risk population. Initially vaccines were highly contaminated by egg derived components and were highly pyrogenic and lacking in efficacy. To overcome problems many different purification techniques and methods for purification of influenza virus were tested. Veerarghavan and Sreevalsan (1961) compared several different methods of concentration and purification of influenza virus and found that two cycles of aluminium phosphate treatment was the best method, followed by ultracentrifugation, erythrocyte adsorption, single cycle aluminium phosphate treatment and finally the zinc oxide method. Early attempts of small scale chromatographic purification of influenza virus on calcium phosphate and on agarose-gel columns were reported in 1960s (Pepper, 1967) but infectivity of the virus was not determined during the experiment. Wallis et al (1972) have shown that influenza virus can be adsorbed on insoluble polyelectrolytes at low pH values under 6.0 and subsequently eluted at high pH. Since influenza virus is pH sensitive and infectivity of purified virus was not determined, applicability of this method for purification of infective influenza virus is not known. Polyethylene glycol precipitation, followed by gel permeation chromatography through controlled pore glass beads was also tested for influenza purification and compared to density gradient procedures (Heyward et al., 1977). The time required for density gradient procedures was tenfold greater than for combination of precipitation and gel permeation chromatography and the purity of virus obtained by both methods was similar. Sagar et al. (1980) used positively charged Zeta Plus filters for influenza virus concentration but for purification further steps are needed as suggested by authors.
U.S. Pat. No. 3,632,745 describes the purification of influenza viruses by dialysing a suspension of the virus against water containing bivalent metallic cations e.g. magnesium. U.S. Pat. Nos. 3,485,718 and 3,547,779 both disclose the use of barium sulphate in the purification of influenza virus. U.S. Pat. No. 3,478,145 discloses the use of calcium phosphate in the purification of influenza virus. U.S. Pat. No. 3,874,999 describes a process consisting of different centrifugation steps, precipitation (with MgSO4), ultra filtration and dialysis. EP-A-0171086 disclose a method for purification of influenza virus with column chromatography using a sulphuric acid ester of cellulose or of a crosslinked polysaccharide. The patent application discloses a purification of influenza virus only from allantoic fluid and does not address virus recovery in terms of virus infectivity which remains unknown. After adsorption of the virus to the column and a washing step, the virus is eluted from the column with buffer containing high NaCl concentration (1.49 M) which influences virus infectivity and lowers recovery of infectious virus. The method is based on a particle support exhibiting low dynamic binding capacity for virus particles which in addition strongly depends on the flow rate. These properties influences down stream process significantly, since large column dimensions are needed and only a low flow rate can be used, all resulting in rather low productivity of the process.
U.S. Pat. No. 6,008,036 discloses a general method for purifying viruses from a cell line culture by chromatography. The disclosed method comprises an ion exchange chromatography step followed by a cation exchange chromatography step and optionally a metal-binding affinity chromatography step.
A major breakthrough in influenza purification was the development of the zonal ultracentrifuge (Reimer et al., 1966). This technology revolutionized the purification process and industrial production of influenza and also other viral vaccines. Up to date, it remains the basis for the down stream process of influenza virus vaccines. Usually purification of influenza virus from allantoic fluids consists of clarification by centrifugation, concentration by ultra filtration and purification by ultra centrifugation. These current methods for the purification of influenza viruses are cumbersome and do not easily allow fast large scale production of virus stocks for vaccines of requested purity.
New influenza epidemics and pandemic are likely to occur in the future and current egg-based vaccine production technology seems to be unable to respond to a pandemic crisis. Thus, a system that can rapidly produce new influenza vaccine is needed. One such approach is a cell culture-based process which can be easily scaled up. But the existing purification of influenza virus vaccine cannot follow this demand. Recently, scalable influenza virus purification process comprising of depth filtration, inactivation, ultra filtration and gel filtration was also described (Prasad Nayak et al, 2005). Since virus was inactivated, applicability of this process for purification of infective influenza virus remains unknown.
Thus, there is still a need for a fast, scalable, efficient and inexpensive down stream process to purify influenza viruses for vaccines or any other application requiring pure, immunogenic and/or infective virus. The present invention discloses such a process.